Microtubules regulate disassembly of epithelial apical junctions

Background  

Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion.     

L-Glutamine ameliorates acetaldehyde-induced increase in paracellular permeability in Caco-2 cell monolayer

Role of L-glutamine in the protection of intestinal epithelium from acetaldehyde-induced disruption of barrier function was evaluated in Caco-2 cell monolayer. L-Glutamine reduced the acetaldehyde-induced decrease in transepithelilal electrical resistance and increase in permeability to inulin and lipopolysaccharide in a time- and dose-dependent manner; d-glutamine, L-aspargine, L-arginine, L-lysine, or L-alanine produced no significant protection. The glutaminase inhibitor 6-diazo-5-oxo-L-norleucine failed to affect the L-glutamine-mediated protection of barrier function. L-Glutamine reduced the acetaldehyde-induced redistribution of occludin, zonula occludens-1 (ZO-1), E-cadherin, and beta-catenin from the intercellular junctions. Acetaldehyde dissociates occludin, ZO-1, E-cadherin, and beta-catenin from the actin cytoskeleton, and this effect was reduced by L-glutamine. L-Glutamine induced a rapid increase in the tyrosine phosphorylation of EGF receptor, and the protective effect of L-glutamine was prevented by AG1478, the EGF-receptor tyrosine kinase inhibitor. These results indicate that L-glutamine prevents acetaldehyde-induced disruption of the tight junction and increase in the paracellular permeability in Caco-2 cell monolayer by an EGF receptor-dependent mechanism.     

16,16-dimethyl prostaglandin E2 modulation of endothelial monolayer paracellular barrier function

Prostaglandins prevent gastrointestinal mucosal injury and promote healing following mucosal injury by various noxious agents. Preservation or repair of microvascular function appears to be crucial in these processes. The processes involved in prostaglandin-mediated repair and preservation of endothelial function are unclear. In the present study, we investigated the role of prostaglandins on endothelial paracellular barrier function using the filter-grown bovine aortic endothelial cell monolayers. Endothelial paracellular barrier function was assessed using a paracellular marker, mannitol. Prostaglandin analogs 16,16-dimethyl prostaglandin E₂ (DMPGE₂) and prostaglandin I₂ (PGI₂) caused an enhancement of endothelial monolayer paracellular barrier function as evidenced by a dose-dependent decrease in endothelial paracellular permeability. DMPGE₂ induced enhancement of endothelial paracellular barrier function correlated directly with increasing intracellular cAMP levels. Agents which increase intracellular cAMP levels at different stages of cAMP amplification cascade including phosphodiesterase inhibitor (3-isobutyl-1 methylxanthine [IBMX]), membrane permeable cAMP (8-bromo cAMP), and adenylate cyclase activators (isoproterenol and forskolin) also produced enhancement in endothelial paracellular barrier function. DMPGE₂ enhancement of paracellular barrier function correlated with dense accumulation of actin microfilaments near the intercellular junctions. IBMX, isoproterenol, forskolin, and 8-bromo cAMP also produced similar changes in endothelial actin microfilaments. Cytochalasin B prevented the DMPGE₂ enhancement of paracellular barrier function. Indomethacin (INDO), a cyclooxygenase inhibitor, caused a dose-dependent increase in endothelial paracellular permeability. Pharmacologic doses of INDO resulted in condensation and disruption of actin microfilaments with formation of large paracellular openings or gaps between the adjacent cells. Pretreatment of endothelial monolayers with DMPGE₂ prevented INDO-induced disturbance of actin microfilaments and paracellular barrier function. IBMX, isoproterenol, forskolin, and 8-bromo cAMP also prevented INDO-induced changes in actin microfilaments and paracellular barrier function. These findings indicate that DMPGE₂ has a paracellular barrier enhancing effect on filter-grown endothelial monolayers. This effect appears to be mediated through intracellular cAMP and actin microfilaments. © 1996 Wiley-Liss, Inc.     

Effects of colchicine and cytochalasin B on hypertonicity-induced changes in toad urinary bladder

Summary Coincident with an increase in the water permeability of toad urinary bladder induced by serosal hypertonicity, a transformation of the ridge-like surface structures of the granular cells into individual microvillous structures occurs. This study was initiated to establish whether the transformation is mediated by the cytoskeletal network and, thus, can be prevented by disruption of microtubulemicrofilament function with colchicine or cytochalasin B (CB). Scanning electron microscopy revealed the characteristic branching ridges on granular cells of control bladder incubated with colchicine or CB. In contrast, transformation of ridges to discrete microvilli was observed in experimental bladders exposed to serosal hypertonicity alone or in combination with either colchicine or CB. These results suggest that the mechanism underlying hypertonicity-induced surface changes which are associated with increased water permeability does not involve either microtubules or microfilaments.     

Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules.

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.     

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.     

Microtubule Integrity Is Necessary for the Epithelial Barrier Function of Cultured Thyroid Cell Monolayers

Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm · cm²). Colchicine (1 μM) caused a progressive fall in electrical resistance to <10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.     

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r) ) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton andZO-1. Thesealterations were abolished byinhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barriercompromise, suggesting a therapeutic potential of statins in intestinal infections.     

Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in monolayers of intestinal epithelial cell

The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H(2)O(2), Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and beta-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H(2)O(2) and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H(2)O(2) alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells.     

Contribution of the Cytoskeleton to the Compressive Properties and Recovery Behavior of Single Cells

The cytoskeleton is known to play an important role in the biomechanical nature and structure of cells, but its particular function in compressive characteristics has not yet been fully examined. This study focused on the contribution of the main three cytoskeletal elements to the bulk compressive stiffness (as measured by the compressive modulus), volumetric or apparent compressibility changes (as further indicated by apparent Poisson's ratio), and recovery behavior of individual chondrocytes. Before mechanical testing, cytochalasin D, acrylamide, or colchicine was used to disrupt actin microfilaments, intermediate filaments, or microtubules, respectively. Cells were subjected to a range of compressive strains and allowed to recover to equilibrium. Analysis of the video recording for each mechanical event yielded relevant compressive properties and recovery characteristics related to the specific cytoskeletal disrupting agent and as a function of applied axial strain. Inhibition of actin microfilaments had the greatest effect on bulk compressive stiffness (∼50% decrease compared to control). Meanwhile, intermediate filaments and microtubules were each found to play an integral role in either the diminution (compressibility) or retention (incompressibility) of original cell volume during compression. In addition, microtubule disruption had the largest effect on the “critical strain threshold” in cellular mechanical behavior (33% decrease compared to control), as well as the characteristic time for recovery (∼100% increase compared to control). Elucidating the role of the cytoskeleton in the compressive biomechanical behavior of single cells is an important step toward understanding the basis of mechanotransduction and the etiology of cellular disease processes.     

Effect of a pore-forming protein derived from Flammulina velutipes on the Caco-2 intestinal epithelial cell monolayer

We have previously found a transepithelial electrical resistance (TEER)-decreasing protein derived from Flammulina velutipes, which was revealed to be identical to flammutoxin (FTX) that is known as a hemolytic pore-forming protein. This protein induced a rapid decrease in TEER and parallel increase in paracellular permeability in the intestinal epithelial Caco-2 cell monolayer without any cytotoxicity. An immunoblotting analysis revealed that the FTX-induced decrease in TEER was accompanied by the formation of a high-molecular-weight complex on the surface of Caco-2 cells. Intracellular Ca(2+) imaging showed that exposure to FTX caused a rapid Ca(2+) influx. It was observed by electron microscopy that FTX induced swelling of microvilli and expansion of the cellular surface. Staining with fluorescent phalloidin showed a marked change to filamentous actin in the FTX-treated cells.These results suggest that TEER reduction could sensitively detect small membrane pore formation by FTX in the intestinal epithelium which causes a morphological alteration and disruption of the paracellular barrier function.     

Microtubules and microfilaments are both responsible for pollen tube elongation in the coniferPicea abies (Norway spruce)

Summary InPicea abies (Norway spruce), microtubules and actin microfllaments both form a dense matrix throughout the tube mainly parallel to the direction of elongation. In these conifer pollen tubes the organization of this matrix is different from that in angiosperms. This study tests our hypothesis that differences in cytoskeletal organization are responsible for differences in tube growth and physiology. Pollen grains were germinated in media containing cytoskeletal disrupters and analyzed for germination, tube length, tube branching, and tip swelling. Disruption of microtubules significantly inhibits tube elongation and induces tube branching and tip swelling. Tip swelling is probably caused by disruption of the microtubules in the tip that are perpendicular to the direction of elongation. Confocal microscopy indicates that colchicine and propyzamide cause fragmentation of microtubules throughout the tube. Oryzalin and amiprophosmethyl cause a complete loss of microtubules from the tip back toward the tube midpoint but leave microtubules intact from the midpoint back to the grain. Disruption of microfilaments by cytochalasins B and D and inhibition of myosin by N-ethylmaleimide or 2,3-butanedione monoxime stops tube growth and inhibits germination. Microfilament disruption induces short branches in tubes, probably originating from defective microfilament organization behind the tip. In addition, confocal microscopy coupled with microinjection of fluorescein-labeled phalloidin into actively growing pollen tubes indicates that microfllament bundles extend into the plastid-free zone at the tip but are specifically excluded from the growing tip. We conclude that microtubules and microfilaments coordinate to drive tip extension in conifer pollen tubes in a model that differs from angiosperms.     

Larazotide acetate promotes tight junction assembly in epithelial cells

Tight junctions (TJ) control paracellular permeability and apical-basolateral polarity of epithelial cells. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. TJ formation is dependent on E-cadherin-mediated cell-cell adhesion and actin rearrangement, and is regulated by the Rho family GTPase and aPKC signaling pathways. Larazotide acetate, an 8-mer peptide and TJ modulator, inhibits TJ disassembly and dysfunction caused by endogenous and exogenous stimuli in intestinal epithelial cells. Here, we examined the effect of larazotide acetate on de novo TJ assembly using 2 different model systems. In MDCK cells, larazotide acetate promoted TJ assembly in a calcium switch assay. Larazotide acetate also promoted actin rearrangement, and junctional distribution of zonula occludens-1 (ZO-1), occludin, claudins, and E-cadherin. Larazotide acetate promoted TJ maturation and decreased paracellular permeability in "leaky" Caco-2 cells. Taken together, our data indicate that larazotide acetate enhances TJ assembly and barrier function by promoting actin rearrangement and redistribution of TJ and AJ proteins.     

Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability

The effects of physiologically relevant increase in temperature (37-41 degrees C) on intestinal epithelial tight junction (TJ) barrier have not been previously studied. Additionally, the role of heat-shock proteins (HSPs) in the regulation of intestinal TJ barrier during heat stress remains unknown. Because heat-induced disturbance of intestinal TJ barrier could lead to endotoxemia and bacterial translocation during physiological thermal stress, the purpose of this study was to investigate the effects of modest, physiologically relevant increases in temperature (37-41 degrees C) on intestinal epithelial TJ barrier and to examine the protective role of HSPs on intestinal TJ barrier. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro intestinal epithelial model system to assess the effects of heat exposure on intestinal TJ barrier. Exposure of filter-grown Caco-2 monolayers to modest increases in temperatures (37-41 degrees C) resulted in a significant time- and temperature-dependent increases in Caco-2 TJ permeability. Exposure to modest heat (39 or 41 degrees C) resulted in rapid and sustained increases in HSP expression; and inhibition of HSP expression produced a marked increase in heat-induced increase in Caco-2 TJ permeability (P < 0.001). Heat exposure (41 degrees C) resulted in a compensatory increase in Caco-2 occludin protein expression and an increase in junctional localization. Inhibition of HSP expression prevented the compensatory upregulation of occludin protein expression and produced a marked disruption in junctional localization of occludin protein during heat stress. In conclusion, our findings demonstrate for the first time that a modest, physiologically relevant increase in temperature causes an increase in intestinal epithelial TJ permeability. Our data also show that HSPs play an important protective role in preventing the heat-induced disruption of intestinal TJ barrier and suggest that HSP mediated upregulation of occludin expression may be an important mechanism involved in the maintenance of intestinal epithelial TJ barrier function during heat stress.     

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.     

TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.     

Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase

The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 microg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg(2+)-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg(2+)-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.     

Glutamine supplementation attenuates ethanol-induced disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier dysfunction and fatty liver in mice

Previous in vitro studies showed that glutamine (Gln) prevents acetaldehyde-induced disruption of tight junctions and adherens junctions in Caco-2 cell monolayers and human colonic mucosa. In the present study, we evaluated the effect of Gln supplementation on ethanol-induced gut barrier dysfunction and liver injury in mice in vivo. Ethanol feeding caused a significant increase in inulin permeability in distal colon. Elevated permeability was associated with a redistribution of tight junction and adherens junction proteins and depletion of detergent-insoluble fractions of these proteins, suggesting that ethanol disrupts apical junctional complexes in colonic epithelium and increases paracellular permeability. Ethanol-induced increase in colonic mucosal permeability and disruption of junctional complexes were most severe in mice fed Gln-free diet. Gln supplementation attenuated ethanol-induced mucosal permeability and disruption of tight junctions and adherens junctions in a dose-dependent manner, indicating the potential role of Gln in nutritional intervention to alcoholic tissue injury. Gln supplementation dose-dependently elevated reduced-protein thiols in colon without affecting the level of oxidized-protein thiols. Ethanol feeding depleted reduced protein thiols and elevated oxidized protein thiols. Ethanol-induced protein thiol oxidation was most severe in mice fed with Gln-free diet and absent in mice fed with Gln-supplemented diet, suggesting that antioxidant effect is one of the likely mechanisms involved in Gln-mediated amelioration of ethanol-induced gut barrier dysfunction. Ethanol feeding elevated plasma transaminase and liver triglyceride, which was accompanied by histopathologic lesions in the liver; ethanol-induced liver damage was attenuated by Gln supplementation. These results indicate that Gln supplementation ameliorates alcohol-induced gut and liver injury.     

Cytoskeletal events underlying dendrite formation by cultured pigment cells

In contrast to neurite outgrowth, pigment cell dendrite formation is relatively unstudied. Keratinocyte-conditioned medium (KCM) induces a striking dendricity in human melanocytes and B16 melanoma cells that is detectable within 30 min, maximal in 24–48 hr, and quantifiable by computerized image analysis. Cyto-chalasin B (CB), known to disrupt actin microfilaments, completely blocks dendrite formation if added to cultures before or with KCM. This effect is rapidly reversible, and dendrites appear within 1 hr after refeeding with KCM alone. In contrast, CB treatment fails to disrupt existing dendrites previously induced by KCM. Agents known to cause microtubule disassembly (colchicine, nocodazole, or vinblastine) do not inhibit dendrite formation if added before or with KCM. In contrast, these agents disrupt established dendrites Inhibition of protein synthesis with cycloheximide or actinomycin D completely blocks dendrite formation, but if cultures are provided fresh KCM lacking protein synthesis inhibitors, dendrites reappear within 24 hr. Actin microfilaments visualized with a monoclonal antibody or rhodamine-phalloidin are poorly organized in untreated cells, but form numerous fibers localized along dendrites in KCM-treated cells. Microtubules visualized with a monoclonal anti-tubulin antibody are localized in the center of dendrites. These cytoskeletal changes occur without altering β actin or β tubulin mRNA levels. Taken together, these data implicate actin microfilaments in dendrite outgrowth, but not in maintenance, and conversely microtubules in dendrite maintenance but not in formation. These keratinocyte-induced changes involving β actin and β tubulin polymerization appear to require both new protein synthesis and post-translational regulation. The observed similarities between melanocytes and other neural crest-derived cells suggest that cutaneous pigment cells might serve as an alternative model for studies of neurite outgrowth. © 1992 Wiley-Liss, Inc.     

Difructose anhydride III and sodium caprate activate paracellular transport via different intracellular events in Caco-2 cells

A nondigestible disaccharide, difructose anhydride (DFA) III, is known to activate calcium transport via tight junctions (TJs); however, the characteristics of and mechanisms for the increase in paracellular transport induced by DFAIII have not been clarified. We compared the effect of DFAIII with that of sodium caprate (C10), a well-known enhancer of TJ permeability, on the changes in TJ proteins, transport of paracellular markers, and effects of nine cellular signaling blockers using Caco-2 monolayers. The addition of DFAIII (0-100mmol/L) and C10 (0-10mmol/L) to the apical medium of the Caco-2 monolayers dose-dependently decreased transepithelial electrical resistance (TER), which is an indicator of TJ permeability. The reduction with C10 was much faster than that with DFAIII. Transport of the paracellular markers of various molecular weights (182-43,200) was elevated by the addition of 100mmol/L DFAIII and 10mmol/L C10. The transport rates were much in the presence of C10 than of DFAIII, while the reduction in TER by two treatments was similar (from 1000 to 300Omega cm(2)). Treatment with DFAIII and C10 changed the distribution of actin filament and claudin-1, but not occludin, junctional adhesion molecule-1, or zonula occludens-1; however, alterations in the patterns of the TJ proteins differed according to treatment. An inhibitor of myosin light chain kinase and a chelator of intracellular calcium ion ([Ca(2+)](i)) attenuated the TER reduction by C10, but not by DFAIII. These data demonstrate that the increase in TJ permeability induced by DFAIII results from the alterations to actin and claudin-1 via [Ca(2+)](i)-independent mechanisms.